Recombinant Polypeptides for Diagnosing Infection with Trypanosoma Cruzi

ABSTRACT

Recombinant polypeptides are disclosed that are useful for diagnosing American trypanosomiasis, or Chagas disease, a disease caused by the infectious agent  Trypanosoma cruzi.  Preferably, DNA sequences encoding the recombinant proteins are placed in plasmid vectors to be expressed in an organism.

This application claims priority from U.S. Provisional Application No.60/430,654, filed Dec. 4, 2002, hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to recombinant polypeptides that areuseful for diagnosing American trypanosomiasis, or Chagas disease.Chagas disease is caused by the infectious agent Trypanosoma cruzi. Moreparticularly, the invention relates to specific combinations ofrecombinant T. cruzi polypeptides, synthesized using genetic engineeringtechniques, and to constructs and processes for producing therecombinant polypeptides, and to an assay and kit for detecting T. cruziinfection which employs the recombinant polypeptides.

2. Background

Chagas disease is a zoonosis caused by the protozoan parasite,Trypanosoma cruzi. This organism is primarily transmitted throughcontact with its triatomine insect vectors, but transmission bytransfusion of contaminated blood and congenital transmission also areimportant. Historically Chagas disease has been a public health problemin all of Latin America, with the exception of the Caribbean nations.The World Health Organization estimates that 16-18 million persons arechronically infected with T. cruzi, and that 45,000 deaths occur eachyear due to the illness. Infection with T. cruzi is life-long andspecific drug treatment lacks efficacy and often causes serious sideeffects. Ten to thirty percent of T. cruzi-infected persons developchronic symptomatic Chagas disease, and the burden of disability andmortality in the endemic countries is enormous.

An estimated 80,000 to 100,000 T. cruzi-infected persons now live in theUnited States. These immigrants pose a risk for transfusion-associatedtransmission of the parasite here and in other countries to which LatinAmericans have emigrated. Eight such cases have been reported in theUnited States, Canada, and Europe, all of which occurred inimmunosuppressed patients in whom acute T. cruzi infection was diagnosedbecause of the fulminant course of the illness. Most transfusions aregiven to immunocompetent patients in whom acute Chagas disease would bea mild illness, and thus it is reasonable to assume that many otherundetected instances of transfusion-associated transmission of T. cruzihave occurred in the United States and other industrialized nations. Thequestion of whether blood donated in the United States should bescreened serologically for antibodies to T. cruzi has been consideredfor at least a decade by both public and private entities involved inblood banking. A panel of experts convened in early 2000 by the AmericanRed Cross to consider this issue recommended unanimously that our bloodsupply be screened serologically. Implementation of such arecommendation, however, is not an option currently because no test forT. cruzi infection has been cleared by the FDA for screening donatedblood.

Diagnosis of T. cruzi infection presents problems. Demographic andclinical data are suggestive at best. Parasitologic tests, e.g.,xenodianosis, hemoculture and PCR are insensitive. Other serologic testsare generally insensitive and lack specificity, as false positivereactions often occur with specimens from patients having infectiousdiseases, such as leishmaniasis, syphilis, or malaria; autoimmunediseases; and other parasitic and non-parasitic illinesses.

Such conventional tests include indirect immunofluorescence (DF),indirect hemagglutination (IHA), and complement fixation (CF) tests, aswell as enzyme-linked immunosorbent assays (ELISA or EIA). Due to thelack of sensitivity and specify of the three commonly used assays, whena sample has a positive result from any, the blood must be discarded.Table I shows that in a major Brazilian blood bank (Hemocentro, SãoPaulo, Brazil), up to 3.43% of blood donations fall into this category.

TABLE I IIF IHA CF % w/ Results + + + 0.68% + − + 0.71% + + − − + + + −− 2.04% − + − − − + TOTAL: 3.43%

Commercially available ELISAs include lysate-based tests such as theChagas Enzyme Immunoassay (EIA), available from Abbott Laboratories ofAbbott Park, Ill. (the subject of FDA 510(k) Premarket Notification No.K933716, herein incorporated by reference in its entirety); the Chagas'IgG ELISA, available from Meridian Bioscience, Inc. of Cincinnati, Ohio,and its predecessor, Gull Laboratories (the subject of FDA 510(k)Premarket Notification No. K911233, herein incorporated by reference inits entirety); and the Chagas' kit (EIA method), available from HemagenDiagnostics, Inc., of Waltham, Mass. (the subject of FDA 510(k)Premarket Notification No. K930272, herein incorporated by reference inits entirely). However, because these tests have less than optimalsensitivities and specificities, their use for screening donated bloodwould fail to detect some T. cruzi-infected units and also would causesubstantial numbers of otherwise usable units to be discardedneedlessly.

One of the present inventors has previously developed a radioimmuneprecipitation assay (RIPA), described in Kirchhoff L V, Gam A A, GusmaoR D, Goldsmith R S, Rezende J M, Rassi A. “Increased specificity ofserodiagnosis of Chagas' disease by detection of antibody to the 72 and90 kDa glycoproteins of Trypanosoma cruzi.” J Infect Dis1987;155:561-564, herein incorporated by reference in its entirety. Thistest is considered the benchmark against which other tests are measured,and it is the only current option for confirmatory testing in the UnitedStates. Unfortunately, the RIPA costs $175 per assay, and at that price,screening the approximately 13 million units of blood donated each yearwould cost over $2 billion.

Therefore, the present inventors have further developed recombinantassays for detection of T. cruzi infection. A typical recombinantpolypeptide and method for assaying is described by them in U.S. Pat.No. 5,876,734, No. 6,228,601, and PCT Publication No. WO 95/25797, eachof which is herein incorporated by reference in its entirety. Suchassays for T. cruzi infection based on recombinant antigens, in contrastto those utilizing native antigens (e.g., the conventional lysate-basedassays), as discussed above, will be more accurate, i.e., thesensitivity and specificity will be higher.

Furthermore, the recombinant assays of the invention presentmanufacturing advantages over the materials for the RIPA andconventional tests. Once the molecular biology has been completed, therecombinant antigens are produced in Escherichia coli, thus eliminatingcompletely any biohazard associated with growing the parasites in liquidculture. This is a substantive advantage, as many cases oflaboratory-acquired T. cruzi infection have been reported. Additionally,recombinant antigens produced in E. coli are much easier to purify,quantitate, and standardize than antigen lysates produced in liquidcultures of parasites, thus facilitating the manufacture of a consistentproduct and simplifying compliance with governmental regulations. Afinal advantage lies in the fact that several of the recombinantproteins presented in this application are comprised of two to fourdistinct protein segments derived from separate T. cruzi genes. This useof hybrid recombinant proteins also facilitates manufacture of an assayin that several antigenically distinct proteins are obtained in a singlepurification, quantitation, and standardization run.

SUMMARY OF THE INVENTION

The present invention utilizes recombinant proteins for detecting T.cruzi infected blood. The invention utilizes specific polypeptidesequences that correspond to fusion proteins FP3, FP4, FP5, FP6, FP7,FP8, FP9 and FP 10 as described below. Isolated polynucleotides thatencode the inventive polypeptides according to the present invention arealso utilized, as are cells transformed with a recombinant plasmid thatexpresses a polypeptide according to the invention. The presentinvention is similar to that which is described in U.S. Pat. No.5,876,734, herein incorporated by reference in its entirety. However,the present invention replaces the proteins in the process with therecombinant proteins of this invention to achieve similar or superiorresults.

The present invention also provides a method for detecting the presenceof antibodies to T. cruzi in an individual, comprising the steps ofcontacting a putative anti-T. cruzi antibody-containing sample from anindividual with a polypeptide according to the invention that istypically attached or conjugated to a carrier molecule or attached orconjugated to a solid phase; allowing anti-T. cruzi and other antibodiesin said sample to bind to said polypeptide; washing away unbound anti-T.cruzi antibodies; and adding a compound that enables detection of theanti-T. cruzi antibodies which are specifically bound to thepolypeptide. The compound that enables detection of the anti-T. cruziantibodies may be selected from the group consisting of a colorometricagent, a fluorescent agent, a chemiluminescent agent and aradionucleotide.

Also provided in accordance with the present invention is a kit fordiagnosing the presence of anti-T. cruzi antibodies in a sample,comprising a container in which a polypeptide according to the inventionis attached or conjugated to a carrier molecule or attached orconjugated to a solid phase; and directions for carrying out the methodaccording to the invention. The kit additionally may comprise acontainer of a compound that binds to anti-T. cruzi antibodies and thatrenders said antibodies detectable.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a description of the prior art.

FIG. 1 a-1 h are schematic representations of the recombinant proteinsutilized in the invention.

FIG. 2 is a bar graph showing reactivity of various blood specimens withrecombinant proteins used alone or in combination as target antigens inELISAs.

DETAILED DESCRIPTION OF THE INVENTION

FIGS. 1 a-1 h represent the recombinant proteins of the invention, withthe various letters indicating known protein sequences, as follows. TheFigs. are schematic diagrams of the recombinant T. cruzi proteins,comprised of segments A through L. Solid segments (A, C, D, F, H, I, andK) represent nonrepetitive proteins having amino acid sequences that areunrelated to each other. Saw-tooth segments (B, E, G, J, and L)represent repetitive proteins having amino acid sequences that areunrelated to each other and unrelated to those of the nonrepetitiveproteins. The relative sizes and numbers of repeats in the repetitiveproteins are roughly represented in the Figs. The sizes and shapes ofthe nonrepetitive segments bear no relation to the actual proteins.

The following information refers to FIGS. 1 and 1 a-1 h in which therecombinant proteins Ag15, FP3, FP4, FP5, FP6, FP7, FP8, FP9 and FP10are depicted schematically. These proteins are derived from T. cruzi,the protozoan parasite that causes Chagas disease, and are formed fromof proteins A through L as indicated, and defined herein. There are nosubstantive amino acid similarities among proteins A through L.Similarly there are no substantive DNA sequence similarities among thesegments that encode proteins A through L. The T. cruzi DNA sequencesthat encode proteins A through L were cloned in combination into pGEXand pET plasmid vectors, such as pET-32a. Strains of Escherichia coliwere transfected with the recombinant vectors bearing the T. cruzi DNAsequences, and the bacteria were incubated in liquid culture underconditions favoring synthesis of the recombinant proteins. The latterproteins were subsequently affinity-purified and then used as targetantigens in ELISAs. ELISAs in which proteins Ag15, FP3, FP4, FP5, FP6,FP7, FP8, FP9, and FP10, alone or in combination are employed as targetantigens are useful as sensitive and specific detectors of anti-T. cruziantibodies in blood specimens obtained from persons who are chronicallyinfected with this parasite. The detection of such antibodies is theprimary means of identifying persons who are chronically infected withT. cruzi.

The following paragraphs contain information relating to the naming,localization, and function of proteins A through L, as well as thecorresponding GenBank accession numbers of the sequences to which theyare related and relevant publications.

It should be noted that the T. cruzi gene segments that encode proteinsegments A through L generally are shortened versions of the nativecoding regions. In this context, the constructs that encode singlesegments (i.e., FP5 and FP9), as well as all the others that encode morethan one segment, are all unique, because, even if the individualcomponents from which the various recombinant proteins of this inventionare known, the segments of the invention have not been combinedpreviously as described herein.

Protein AB. This hybrid recombinant protein, also designated Ag15 [SEQID NO. 2] in FIG. 1, is derived from the TCR27 gene of T. cruzi [SEQ IDNO. 1]. Protein A is the amino terminal nonrepetitive portion of theTCR27 protein, and Protein B is comprised of approximately 18 of the 14amino acid repeats that make up the central portion of the TCR27protein. The two native TCR27 genes sequenced contained approximately 69and 105 of the 14-amino acid repeats.

Nucleotide sequence data that include the Ag15 DNA sequence weredeposited with GenBank and EMBL databases by Keiko Otsu, John E.Donelson, and Louis V. Kirchhoff with the accession number L04603 andare described in U.S. Pat. No. 5,876,734 and No. 6,228,601, issued toLouis V. Kirchhoff and Keiko Otsu (each of which is herein incorporatedby reference in its entirety). These references also present DNA andinferred protein sequences that include the Ag15 DNA and inferredprotein sequences. The Ag15 DNA and inferred protein sequences areadditionally presented in Otsu K, Donelson J E, Kirchhoff L V.“Interruption of a Trypanosoma cruzi gene encoding a protein containing14-amino acid repeats by targeted insertion of the neomycinphosphotransferase gene.” Mol Biochem Parasitol 1993;57:317-330, hereinincorporated by reference in its entirety.

Protein C. This is a calcium binding protein of T. cruzi, initiallycalled 1F8 and later designated the flagellar calcium binding protein(FCaBP) [SEQ ID NO 4]. The accession number of the original 1F8 DNAsequence [SEQ ID NO 3] deposited in GenBank is K03278. The Protein C DNAand inferred protein sequences are presented in Gonzalez A, Lerner T J,Huecas M, Sosa-Pineda B, Nogueira N, Lizardi P M. “Apparent generationof a segmented mRNA from two separate tandem gene families inTrypanosoma cruzi.” Nucleic Acids Res 1985;13(16):5789-804, hereinincorporated by reference in its entirety.

FIG. 1 a shows a first protein (FP3) [SEQ ID NO. 22] in accordance withthe invention. Specifically, FP3 corresponds essentially to thecombination of Ag15 (FIG. 1), and by Protein C. The DNA sequenceencoding FP3 [SEQ ID NO 21], also essentially corresponds to thesequences coding for Ag15 and Protein C.

Protein D. This is the protein core of a surface glycoprotein of T.cruzi that is referred to as GP72 [SEQ ID NO 6]. The accession number ofthe original gp72 DNA sequence [SEQ ID NO 5] deposited in GenBank isM65021. The Protein D DNA and inferred protein sequences are presentedin Cooper R, Inverso J A, Espinosa M, Nogueira N, Cross G A.“Characterization of a candidate gene for GP72, an insect stage-specificantigen of Trypanosoma cruzi.” Mol Biochem Parasitol 1991;49(1):45-59,herein incorporated by reference in its entirety.

FIG. 1 b shows a second protein (FP4) [SEQ ID NO 8] in accordance withthe invention. The DNA sequence [SEQ ID NO 7] that encodes Protein DABCwhich is a single continuous coding region, essentially corresponds tothe DNA sequences from which it was constructed.

Protein E. This is a segment of the flagellar repetitive protein (FRA)[SEQ ID NO 10] of T. cruzi comprised of approximately nine repeatsconsisting of 68 amino acids each, shown as FIG. 1 c (FP5). Theaccession number of the original Protein E DNA sequence [SEQ ID NO 9]deposited in GenBank is J04015. The Protein E DNA and inferred proteinsequences are presented in Lafaille J J, Linss J, Krieger M A,Souto-Padron T, de Souza W, Goldenberg S. “Structure and expression oftwo Trypanosoma cruzi genes encoding antigenic proteins bearingrepetitive epitopes.” Mol Biochem Parasitol 1989; 35(2):127-136, hereinincorporated by reference in its entirety.

Protein FGH. This is a protein [SEQ ID NO 12] encoded by a modifiedversion of the T. cruzi TCR39 gene that was artificially constructed[SEQ ID NO 11], shown as FIG. 1 e (FP7). The modification entailedreducing the length of the central portion of the TCR39 gene thatencodes the 12-amino acid repeats. Protein F is the amino terminalnonrepetitive segment of the TCR39 protein. Protein G is comprised ofapproximately 13 of the 12-amino acid repeats that make up the centralportion of the TCR39 protein. Protein H is the carboxy terminalnonrepetitive segment of the TCR39 protein. The accession number of theoriginal, i.e., the unmodified, Protein FGH DNA sequence deposited inGenBank is U15616. The TCR39 DNA and inferred protein sequences, whichinclude the entire Protein FGH sequences, are presented in Gruber A,Zingales B. “Trypanosoma cruzi: characterization of two recombinantantigens with potential application in the diagnosis of Chagas'disease.” Exp Parasitol 1993;76(1):1-12, herein incorporated byreference in its entirety.

FIG. 1 d shows another hybrid recombinant protein (FP6, Protein FGHE)[SEQ ID NO 14] in accordance with the invention. The DNA sequence thatencodes Protein FGHE [SEQ ID NO 13], which is a single continuous codingregion, essentially corresponds to the DNA sequences from which it wasconstructed.

Protein IJK. This is a protein [SEQ ID NO 16] encoded by a modifiedversion of the T. cruzi shed acute phase antigen (SAPA) gene that wasartificially constructed [SEQ ID NO 15], as shown in FIG. 1 f (FP8). Themodification entailed reducing the length of the central portion of theSAPA gene that consists of 12-amino acid repeats. Protein I is the aminoterminal nonrepetitive segment of the SAPA protein. Protein J iscomprised of approximately nine of the 12-amino acid repeats that makeup the central portion of the SAPA protein. Protein K is the carboxyterminal nonrepetitive segment of the SAPA protein. The accession numberof the original, i.e., the unmodified, Protein IJK DNA sequencedeposited in Gen Bank is J03985. The SAPA DNA and protein sequences,which include the entire Protein IJK sequences, are presented inAffranchino J L, Pollevick G D, Frasch A C C. “The expression of themajor shed Trypanosoma cruzi antigen results from thedevelopmentally-regulated transcription of a small gene family.” FEBSLett 1991;280:316-320, herein incorporated by reference in its entirety.

Protein L. This is a microtubule-associated repetitive protein (MAP)[SEQ ID NO 18] of T. cruzi that is comprised of approximately fiverepeats consisting of 38 amino acids each, as depicted in FIG. 1 g(FP9). The accession number of the original Protein L DNA sequence [SEQID NO 17] deposited in GenBank is S68286. The Protein L DNA and inferredprotein sequences are presented in Kerner N, Liegeard P, Levin M J,Hontebeyrie-Joskowicz M. “Trypanosoma cruzi: antibodies to a MAP-likeprotein in chronic Chagas' disease cross-react with mammaliancytoskeleton.” Experimental Parasitology 1991;73(4):451-459, hereinincorporated by reference in its entirety.

FIG. 1 h shows another hybrid recombinant protein (FP10, Protein IJKL)[SEQ ID NO 20] in accordance with the invention. The DNA sequence thatencodes Protein IJKL [SEQ ID NO 19], which is a single continuous codingregion, essentially corresponds to the DNA sequences from which it Wasconstructed.

Additionally, combinations of the various recombinant proteins depictedin the Figs. may be used. While it is possible to combine one or more ofthe recombinant proteins to form longer recombinant proteins, typicallymore than one recombinant protein is used simultaneously. For example,simultaneous uses of FP4 and FP5, FP5 and FP6, as well as FP4 and FP6,and combinations using more than two recombinant proteins (e.g., FP4,FP6 and FP10) are considered within the scope of the present invention.It is believed that the sensitivity and specificity of the assaysaccording to the invention are sufficient to meet FDA standards forscreening the blood supply of the United States.

Additionally, as described in U.S. Pat. No. 6,228,601 (hereinincorporated by reference in its entirety), polypeptides need notcorrespond exactly over their entire lengths to be considered within thescope of the invention. For example, a wide variety of polypeptideswhich contain at least one epitope embodied in the polypeptides of theinvention can be used in accordance with the present invention. Based onthe nucleotide sequences, polypeptide molecules also can be produced (1)that include sequence variations, relative to the naturally-occurringsequences, (2) that have one or more amino acids truncated from thenaturally-occurring sequences and variations thereof, or (3) thatcontain the naturally-occurring sequences and variations thereof as partof a longer sequence.

In this description, polypeptide molecules in categories (1), (2) and(3) are said to “correspond” to the amino acid sequences of therecombinant proteins of the invention. Such polypeptides also arereferred to as “variants.” The category of variants within the presentinvention includes, for example, fragments and muteins of proteins Athough L, as well as larger molecules that consist essentially at leastone protein sequence A through L, alone or in combination with otherproteins A to L.

In this regard, a molecule that “consists essentially of” protein A toL, alone or in combination with any other proteins A to L, is one thatis immunoreactive with samples from persons infected with T. cruzi, butthat does not react with samples from patients with leishmaniasis,schistosomiasis, and other parasitic and infectious diseases, withsamples from patients with autoimmune disorders and other illnesses, andwith specimens from normal persons.

A “mutein” is a polypeptide that is homologous to the protein to whichit corresponds, and that retains the basic functional attribute—theability to react selectively with samples from persons infected with T.cruzi—of the corresponding region. For purposes of this description,“homology” between two sequences connotes a likeness short of identityindicative of a derivation of the first sequence from the second. Inparticular, a polypeptide is “homologous” to the corresponding proteinif a comparison of amino acid sequences between the polypeptide and thecorresponding region reveals an identity of greater than 40%, preferablygreater than 50% and more preferably 70%. Such sequence comparisons canbe performed via known algorithms, such as those described in Pearson WR, Lipman D J. “Improved tools for biological sequence comparison.” ProcNatl Acad Sci USA 1988;85(8):2444-2448, herein incorporated by referencein its entirety, which are readily implemented by computer.

A fragment of a protein of the invention is a molecule in which one ormore amino acids are truncated from that protein. Muteins and fragmentscan be produced, in accordance with the present invention, by known denovo synthesis techniques.

Also exemplary of variants within the present invention are moleculesthat are longer than a protein of the invention, but that contain theregion or a mutein thereof within the longer sequence. For example, avariant may include a father fusion partner in addition to the proteinof the invention. Such a fusion partner may allow easier purification ofrecombinantly-produced polypeptides. For example, use of aglutathione-S-transferase (26 kilodaltons, GST) fusion partner allowspurification of recombinant polypeptides on glutathione agarose beads.

The portion of the sequence of a such molecule other than that portionof the sequence corresponding to the region may or may not be homologousto the sequence of a protein of the invention.

It will be appreciated that polypeptides shorter than the correspondingprotein of the invention but that retain the ability to reactselectively with samples from persons infected with T. cruzi aresuitable for use in the present invention. Thus, variants may be of thesame length, longer than or shorter than the protein of the invention,and also include sequences in which there are amino acid substitutionsof the parent sequence. These variants must retain the ability to reactselectively with samples from persons infected with T. cruzi.

In one embodiment, the assay of the invention uses FP4 as targetantigen. Table II compares the results obtained by testing 45pre-screened Argentinean specimens in an

TABLE II RIPA + − FP4 ELISA + 9 0 − 0 36FP4 ELISA with those obtained by RIPA testing.

The data in Table II show that in this group of specimens, thesensitivity and specificity of the FP4 ELISA were both 100%

Similarly, the performance of an FP4+FP6 ELISA in comparison to RDA was

TABLE III RIPA + − FP4 + FP6 ELISA + 10 1 − 0 78assessed by testing 89 pre-selected Guatemalan specimens.

The data shown in Table III indicate that in this group of samples, thesensitivity of the FP4+FP6 ELISA was 100% and the specificity was 98.7%.

As shown in FIG. 2, in a FP4+FP6 ELISA, performed using standardprocedures, a group of previously characterized RIPA-positive samplesfrom several Chagas-endemic countries gave a mean reactivity(absorbance) of 2.99. Thus FP4+FP6 is the preferred embodiment among therecombinant proteins tested alone and in combination in that experiment.

It should be apparent that embodiments other than those specificallydescribed above may come within the spirit and scope of the presentinvention, such as recombinant proteins comprised of differentcombinations and/or spatial arrangements of proteins A to L. Hence, thepresent invention is not limited by the above description.

1. A recombinant plasmid vector comprising a DNA sequence which codesfor a recombinant polypeptide corresponding to a polypeptide selectedfrom the group consisting of FP3 [SEQ ID NO 22], FP4 [SEQ ID NO 8], FP5[SEQ ID NO 10], FP6 [SEQ ID NO 14], FP7 [SEQ ID NO 12], FP8 [SEQ ID NO16], FP9 [SEQ ID NO 18] and FP 10 [SEQ ID NO 20].
 2. The plasmid vectorof claim 1, wherein the plasmid vector is selected from the groupconsisting of pGEX and pET plasmid vectors.
 3. The plasmid vector ofclaim 2, wherein the plasmid vector is pET-32a.
 4. The plasmid vector ofclaim 1, wherein the DNA sequence comprises one selected from the groupconsisting of SEQ lD NO 21, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 13, SEQID NO 11, SEQ ID NO 15, SEQ ID NO 17, and SEQ ID NO
 19. 4. An organismtransfected with the plasmid vector of claim
 1. 5. The organism of claim4, wherein the organism is Escherichia coli.
 6. A recombinantpolypeptide comprising a sequence corresponding to one of FP3, FP4, FP6,FP7, FP8 and FP10.
 7. A kit comprising: a first recombinant polypeptidewherein the first recombinant polypeptide is the recombinant polypeptideof claim 6, and a second recombinant polypeptide.
 8. The kit of claim 7,wherein the second polypeptide comprises a sequence corresponding to oneselected from the group consisting of Ag15 [SEQ ID NO 2], FP3, FP4, FP5,FP6, FP7 FP8, FP9 and FP10, wherein the first recombinant polypeptide isdifferent from the second recombinant polypeptide.
 9. The kit of claim7, wherein the first recombinant polypeptide is FP4 and the secondrecombinant polypeptide is FP6.
 10. The kit of claim 7, furthercomprising a third recombinant polypeptide selected from the groupconsisting of Ag15, FP3, FP4, FP5, FP6, FP7 FP8, FP9 and FP10, whereinthe first recombinant polypeptide, the second recombinant polypeptideand the third recombinant polypeptide are different.
 11. The kit ofclaim 10, wherein the first recombinant polypeptide corresponds to FP4,the second recombinant polypeptide corresponds to FP6 and the thirdpolypeptide corresponds to FP10.
 12. A method of detecting the presenceof anti-Trypanosoma cruzi antibodies in a sample from a subject,comprising: (A) contacting the sample with a polypeptide comprising anamino acid sequence selected from the group consisting of FP3, FP4, FP6,FP7, FP8 and FP10 or an immunoreactive fragment thereof, and (B)detecting a specific binding interaction with an antibody in saidsample, wherein the binding interaction comprises a specific bindingbetween antibody in the sample and an epitope contained within the aminoacid sequence set forth in FP3, FP4, FP6, FP7, FP8 and FP10 and whereinsaid specific binding interaction indicates past or present infectionwith Trypanosoma cruzi.
 13. The method of claim 12, wherein thepolypeptide of step A is immobilized on a carrier molecule or a solidphase.
 14. The method of claim 12, wherein the polypeptide of step A hasa sequence obtained from a strain or clone of Trypanosoma cruzi.
 15. Themethod of claim 12, wherein the polypeptide has had one or more aminoacids truncated.
 16. The method of claim 12, wherein the step ofdetecting anti-Trypanosoma cruzi antibodies bound to the immobilizedpolypeptide is carried out by adding at least one compound that detectsthe antibodies.
 17. The method of claim 16, wherein the at least onecompound that enables detection of the anti-Trypanosoma cruzi antibodiesis selected from the group consisting of a colorimetric agent, afluorescent agent, a chemiluminescent agent and a radionucleotide.